The energy-producing organisms such as plants, some algae, and cyanobacteria produce energy not only for themselves but also for almost all other organisms on Earth. The product of photosynthesis is glucose, the immediate energy source of cells. Once photosynthesized, glucose can be used either to make energy for cellular work or converted into complex molecules of starch, oil, and proteins as stored energy, structural components, or molecular machineries. Proteins in cells serve critical functions such as structural components, enzymes, transport proteins, and cellular energy source. The structural components of plant cell wall are made up of 2-10% proteins, and the cell membrane contains 50% proteins. The breakdown of proteins and mobilization of amino acids depend on the physiologic needs of plants under various conditions such as oxidative stress, salinity, seasonal change, and developmental stages.
Plants grown in a medium with high salt concentrations have lower energy levels, are shorter, have smaller biomass, and have a lower protein content in comparison with plants grown in low salt concentrations (Maria et al. 2001). As a case in point, Asish et al. (2005) showed that a salt-sensitive protein SSP-23 degrades at high salt concentration in the mangrove plant species Bruguiera parviflora, and the phenomenon is suggestive of a salt tolerance mechanism, which adjusts for the osmolarity difference by releasing the protein's amino acid content. High-salt medium seems to be a catalyst for protein breakdown like the SSP-23 degradation and its amino acid mobilization.
Plants adjust the level of energy utilization according to their developmental needs. During the winter, energy is reserved in the form of starch, proteins, and amino acids, which are derived from glucose photosynthesized during late summer. In the fresh pith of tobacco plant species Nicotiana tabacum, which represents a slow-growing tissue much like plants in winter months, 88% of the total cellular amino acids are present in a soluble pool with only 12% incorporated into proteins. Conversely the plant's callus, a rapid outgrowth tissue from growing the pith tissue in an artificial nutrient-rich medium, has only 8% of total amino acids in the soluble pool and 92% in proteins. Further, the most abundant amino acids in the soluble pool are glutamine, asparagine, glutamine, and aspartic acid (Kemp et al. 1972). The amino acid reserve of alanine, arginine, and asparagine comes from the nitrogen fixation of ammonia, probably via glutamine (Menegus et al., 1993). These reserve sources of energy and nitrogen-rich compounds are tapped in the spring when apical growth begins.
During the metabolically-dormant winter, plants do not produce much photosynthesized oxygen that can breakdown cell membrane and, thus, release its protein content. For example, some rhizomes (creeping rootstalks) such as P. australis, A. calamus, and S. lacustris keep their membranes intact during the winter. The growth condition that begins in the spring provides a sudden surge in oxygen that can damage cell membrane though the process called lipid peroxidation. Then, membrane-bound proteins are released, which provide an amino acid pool as a source for both energy and for making other proteins needed for growth. However, the rootstalk plant species Iris pseudocorus produces the anti-oxidant enzyme superoxide dismutase (SOD) during the anoxic winter season for stabilizing the oxidant surge in the spring. For those species that do not produce SOD just before spring, the change in season provides a burst of protein pool as a source of energy for the growth requirement.
Under the oxygen deprivation condition called anoxia, plants arrest oxidative phosphorylation and produce only 2 molecules of ATP during fermentation rather 36 ATP under oxygen-using metabolism. In response to anoxic stress, plants adapt by increasing the ATP production rate (Pasteur Effect) although not totally making up for the energy deficit. Under anoxia, the synthesis of plant proteins is inhibited by the destabilization of the protein-producing machineries of polysomes (Baily-Serres, 1990). Some genes turned on by anoxia are metabolism-specific and include alcohol dehydrogenase, pyruvate decarboxylase, enolase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and sucrose synthase (Sachs et al. 1996), and these enzymes speed up the conversion of energy substrates into energy molecules to compensate for the lack of an energetic state during anoxia. With limited protein synthesis during anoxia, growth is impeded in most plant tissues except for the seed leaf (coleoptile) of germinating rice seedlings, which is unique in its fast growing rate under anoxia while other organs of the seedlings are inhibited from growing (Vartapetian et al., 1978). The energy for the coleoptile growth may come from the rice seed covering layer aleurone, which increases its soluble protein pool in addition to its protease concentrations during germination (Miyuki et al., 2002). With the intact rice seed containing 7-9% protein content, the significant pool of amino acids can provide the initial substrates for conversion into sugar by gluconeogenesis.
The growth-inhibiting stress factors mentioned above ultimately deprive plants' ability to make new sources of energy by inhibiting access to either water, sunlight, or oxygen, all necessary components of photosynthesis. If the immediate energy source is not supplied, plants resort to stored sources of energy locked in starch, oil, and proteins as seen under stress conditions. All 18 of the 20 naturally-occurring amino acids are precursors of substrates for gluconeogenesis to make glucose. If plants were engineered to be more efficient at performing gluconeogenesis, specific substrates along the metabolic pathways should be targeted for manipulation. Pyruvate, a substrate situated at a metabolic crossroad, can feed it into fermentation under anaerobic condition or cell respiration under aerobic condition. To divert pyruvate from entering anaerobic or aerobic respiration and, thus, depleting a viable source for glucose synthesis, pyruvate can be induce-metabolized into oxaloacetate by the over production of pyruvate carboxylase, the enzyme for the conversion. The abundance of oxaloacetate, a precursor for gluconeogenesis, may push the reactions forward. Pyruvate-oxaloacetate conversion also may stimulate the conversions of alanine, cysteine, glycine, serine, and threonine amino acids into pyruvate. The remaining 13 amino acids in FIG. 1 also may be stimulated to enter the cycle for the production of oxaloacetate if the accumulated substrate pool were used for phosphoenolpyruvate synthesis by PEPCK (phosphoenolpyruvate carboxylase).
When plants are subjected to various stress and growth-limiting conditions, metabolism is switched from energy storing (anabolism) to energy usage (catabolism). During periods of energy abundance, plants have evolved to store the excess energy in starch, oil, and proteins. When energy input is limited, the stored sources of energy can undergo metabolic interconversion into glucose via gluconeogenesis. Proteins are a major category of biomolecules in plants (corn kernels contain 9-10% proteins), and tapping into methods to convert proteins to sugars provide an added source of accessible substrates for ethanol production. Stress factors can mobilize the stored energy, and plants also can be engineered to actively take the gluconeogenic pathway. However, biomass buildup depends on the constant energy input without excessive external stress. To harvest the most possible sugars, plants may be allowed to build up biomass under nutrient-rich conditions then be subjected to stress just before harvesting, or plants can be engineered to grow normally while metabolizing sugars. How much economically-viable products we can extract from algal biomass depend on the available forms. A major hurdle in biofuel production is yield, and efforts to increase yield has focused on genetic engineering.
The current standard ethanol fermentation uses glucose sugar as feedstock, thus, limiting yield. The U.S. ethanol industry uses mostly corn as a feedstock, which contains 72% starch sugar that must be processed into glucose sugar for ethanol fermentation. Another source of glucose comes from cellulosic materials, which also have to be processed into glucose. Therefore, there still is a need for an alternative and improved production process of ethanol, which does not have all the drawbacks of existing processes. The engineered organisms herein described can use proteins in addition to sugars as carbon sources, therefore, increasing ethanol fermentation yield. Although the sugar glucose is the immediate energy source for cells, they can convert other nutrients like proteins, fats, and carbohydrates into glucose by the metabolic process called gluconeogenesis. The engineered organisms as described herein have enhanced gluconeogenesis pathways that can build up glucose, the feedstock for ethanol fermentation. This system has several advantages over the current ethanol fermentation system including increasing ethanol yield, using proteins and carbohydrates as sources of carbon, and making the clean biofuel production more economical.